1. Field of the Invention
The present invention generally concerns the preparation of gels, typically agarose gels, that are suitable to receive samples upon which electrophoresis is performed.
The present invention particularly concerns an apparatus and a method for the easy simultaneous preparation of multiple trays of gels, typically agarose gels, that are suitable to receive samples upon which electrophoresis is performed.
2. Description of the Prior Art
Agarose gel electrophoresis is commonly used for both analytical and preparative separation of DNA fragments. Standard agarose gels separate DNA fragments from .apprxeq.0.1 to 25 kilobases, or kb, whereas pulsed-field agarose gels resolve molecules from .apprxeq.10 to &gt;2000 kb. Descriptions of standard and pulsed-field agarose gel electrophoresis, as well as parameters affecting resolution of large DNA fragments, are presented in standard textbooks. At least three different protocols employing agarose gels to prepare DNA fragments are known. An agarose gel has the properties of an electric circuit.
One leading protocol for using agarose gel electrophoresis as a simple and highly effective method for the separation, identification and purification of .apprxeq.0. to 25 kb DNA fragments can be divided into three stages. First, a gel is prepared with an agarose concentration appropriate for the size of DNA fragments to be separated. Second, the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation. Third and finally, the gel is stained or, if ethidium bromide has been incorporated into the gel and into the electrophoresis buffer, visualized directly upon illumination with ultraviolet (UV) light.
In order to conduct agarose gel electrophoresis, an agarose gel must be prepared. This preparation is the subject of the present invention.
Preparation typically commences by sealing the edges of a clean, dry, glass plate (or the open ends of the plastic tray supplied with the electrophoresis apparatus) with autoclave tape so as to form a mold. An electrophoresis buffer (usually 1.times.TAE or 0.5.times.TBE) sufficient in amount so as to fill the electrophoresis tank and to prepare a gel is prepared. An amount of powdered agarose is added to a measured quantity of electrophoresis buffer in an Erlenmeyer flask or a glass bottle with a loose-fitting cap. The same batch of electrophoresis buffer is used in both the electrophoresis tank and the gel.
The neck of the Erlenmeyer flask is loosely plugged. The slurry of powdered agarose and buffer is heated in a boiling-water bath or a microwave oven sufficiently so as to permit all of the grains of agarose to dissolve. The dissolved solution is cooled to 60.degree. C., and if desired, ethidium bromide is added.
The liquid is poured into the mold and permitted to cool to produce a gel. The higher the concentration of agarose, the quicker the gel hardens.
In order to produce slits, or wells, in the gel--into which wells samples undergoing electrophoresis will be inserted--a separate comb is typically positioned 0.5-1.0 mm above the glass plate, or base of the mold. The teeth of the comb form a series of linearly aligned wells when the liquid agarose solution is added to the mold. When the comb is properly manually positioned above the plate, or base of the mold, then it is typically supported on its outer teeth so that its inner teeth will be automatically lifted slightly above, and clear of, the plate.
After the gel is completely set both the comb and autoclave tape are carefully removed, and the gel is mounted in the electrophoresis tank. Just enough electrophoresis buffer is added so as to cover the gel to a depth of about 1 mm.
A sample of DNA is mixed with a desired gel-loading buffer. This mixture is slowly loaded into the slits, or wells, of the submerged gel using a disposable micropipette, an automatic micropipettor or a pasteur pipette. Electrophoresis is then performed.
This prior art procedure for the production of a tray of gel, typically an agarose gel, is (i) labor intensive, and thus (ii) expensive, (iii) tedious, and (iv) ill-adapted to efficiencies of scale because a large number of gel trays are roughly more difficult to make than is a single gel tray in proportion to the numbers thereof.
Moreover, the slits, or wells, that are formed in the gel may be of improper or inconsistent depth in accordance that the manual alignment procedure induces errors.
Admittedly, separation by process of electrophoresis of such samples as are later inserted within the slits, or wells, of the gel may accord a certain registration upon viewing (or photographing) that is based on the innate visual properties of the samples. However, it is illogical, and of dubious soundness as a scientific procedure, that successive gels upon which electrophoresis is performed should not be, insofar as is possible, identical. Although exact chemical identity may not be possible between successive gels that are not simultaneously prepared (the present invention will be seen to greatly abet simultaneous preparation of multiple gels), any significant mechanical variation in gel size and thickness, and the locations and depths of the slits (wells) locations in the gels should be, insofar as is possible, strictly avoided.
Presently, the greatest problem occurs with adjusting the depths of the slits should a one, predetermined, slit depth offered by current equipments prove unsatisfactory. Precision machined combs that are matched to gel trays, and which are accurately positionable thereon by action of tongues and grooves, are known. Variable numbers of slits, or wells, may be selected (in accordance that the comb has a greater, or smaller, number of teeth). However, these slits, or wells, are necessarily of differing depths in gels of various thicknesses. Sometimes it is sufficient, and desired, that all slits, or wells, in all of a number of gels should be formed to be at a uniform height, nominally 1 mm, above the base of the tray at their lowermost extension. The present precision gel combs accord this capability. However, it is sometimes desirable to leave a greater thickness below the wells. The present system does not support of creating wells having a variably selectable depth, which depth may, nonetheless to being variably selectable, be uniform from gel tray to gel tray across as large number of gel trays.
Accordingly, it would be desirable if the limitations of the present procedures for the production of agarose gels could be overcome, and if a large number of gel trays complete with precision located and arrayed slits, or wells, could be reliably, easily and inexpensively made. Moreover, it would be desirable if the numbers and three-dimensional positions--especially the extent above gel tray bottom--of the slits, or wells, which are formed in the gel could be, nonetheless to being variably selectable, precisely controllable. "Precisely controllable" means that two or more gel trays might each be made at separate times to custom mechanical characteristics that are effectively identical. For example, it would be useful if, as an arbitrary example, a gel tray having the physical dimensions of 20 cm by 30 cm bed dimension containing a gel of a (relatively thick) 7 mm perforated at exactly 10 slits of 1.0 mm each at positioned 1 cm from a short side wall, each slit to a (relatively high) 1.5 mm above the base of the tray could be repetitively reliably made, and re-made. This precision over a range of dimensions--especially as relate to the extent of the slits above the base of the gel tray--is effectively impossible with present apparatus, and procedures.
The present invention will be seen to concern an apparatus and method for greatly (i) reducing the labor, and thus (ii) reducing the expense, (iii) reducing the tedium, and (iv) enhancing the efficiency of producing large numbers of gel trays by introducing economies of scale to the entire process.